A. Field of the Invention
The present invention relates to a method for DNA comparative analysis in a plurality of samples and a sample preparation method for the DNA analysis.
B. Description of the Prior Art
With the progress of genome analysis, the first stage of the genome project, where the analysis of genome structures by DNA sequencing is the major subject, is going to the end and the genome analysis comes to the second stage of understanding gene functions. The genetic information in genome sequences has to be translated to a protein through mRNA. The genes expressed in a cell at some moment can be determined by detecting mRNAs in the cell. Genetic characteristics of individuals are dependent on various differences in their genome sequences. The analysis of mRNAs in cells or tissues and the comparative analysis of DNA sequences for individual genes are necessary for understanding the gene functions. Especially the analysis of species and amounts of mRNAs in cells is important to know what is going on in the cells. Usually, cDNA (complementary DNA), which is produced by complementary strand extension reactions with a DNA polymerase and a primer hybridizing to each mRNA, is used for the analysis instead of mRNA because mRNAs are easily decomposed by RNase that is in cells.
The scanning of all the cDNAs (or mRNAs) in cells or tissues is called as gene expression profiling. As the size of each cDNA is usually very long to be sequenced or to be analyzed by gel electrophoresis, a part of the sequence is selected as the signature sequence of the cDNA to be analyzed. Each of the signature sequences of cDNAs is amplified and analyzed by gel electrophoresis or by hybridization with a DNA probe array. At first, the signature regions of cDNAs are amplified by PCR (Polymerase Chain Reaction) and then the relative abundance of each signature fragment is analyzed. The key point of the method is how to amplify each of signature regions without losing the relative abundance information. The relative abundance information is frequently lost during the PCR process because the amplification factors of each PCR reaction are dependent on the precise conditions and the sequence of the target cDNA fragment. The PCR amplification for plural of target DNA fragments should be carried out simultaneously to keep the amplification conditions the same. However, it is not so easy because the primers used for amplifying the target DNA fragments frequently interact with each other to produce undesired new DNA fragments which disturb the accuracy and reliability of the gene expression profiling.
The present invention relates to a method for carrying out the simultaneous PCR amplification of various cDNA fragments for quantitative cDNA analysis such as gene expression profiling. The invention also relates to the method for recovering PCR products and the sample preparation for DNA diagnostics. In PCR amplification, two primers are designed to hybridize on the template DNA at predetermined positions. The base sequence of the template DNA sandwiched with the two primers is amplified by repetitive complementary strand extension reactions with the primers. The number of copies of the target DNA fragments increases by several orders of magnitude by PCR. In the case of gene expression profiling, a DNA sample contains various cDNAs fragment species. Many should be analyzed quantitatively. The method requires the PCR amplification of plural of target cDNA fragment species simultaneously. When the PCR amplification of a plurality of DNA fragments or sequences is carried out, artificial fragments are frequently produced through unexpected reactions among primers and DNAs. However, the isolation of the amplified components is labor intensive. Consequently, only one pair of primers is used at a time for PCR amplification. When many target DNA fragment species have to be analyzed, many PCR reactions are required. This is very labor intensive.
On the other hand, the comparative analysis for two or more kinds of DNA fragments is an important subject and is extensively investigated. However, since the amplification rate in PCR depends greatly on the reaction conditions, the comparison of groups of DNA fragments which are obtained under different PCR conditions, namely, groups of DNA fragments which are independently obtained by amplification, has been disadvantageous in that it prohibits quantitative investigation. Factors capable of affecting PCR include the reaction temperature, the base sequences of primers, the amounts of reagents, the kinds and amounts of contaminants, etc. It is considerably difficult to make these factors the same for different reactions.
A PCR technique for quantitative and comparative analysis for one DNA fragment species in various samples such as tissues has recently been developed. This method is called adopter-tagged competitive PCR (ATAC PCR). Now the target of the analysis is the same DNA fragment species in different DNA samples (for example, different sample numbers are used to identify those samples; sample number 1-sample number 9). There are plural of samples containing various DNA species to be compared. The method can carry out comparative analysis of DNA fragment species belonging to different samples by putting tags depending on the samples. The tagging is taken place by changing the lengths of oligomers connected to the DNA fragments as follows. An oligonucleotide having a known base sequence is connected to each end of the DNA fragment species. The known base sequence is composed of a common base sequence for the hybridization of a primer and a tagging base sequence for discriminating the plurality of the samples containing various DNA species. To separate DNA fragment species produced from different samples, the tagging sequences are designed so as that their lengths are different from sample to sample. In ATAC PCR analysis, only one target DNA fragment species in various samples is analyzed at a time. Each sample contains the target DNA fragment sequences at different ratios. The priming site for PCR amplification is also the same for different DNA fragments. The only difference in the targets is the lengths of the tagging sequence region. Consequently, all the target DNA fragments can be amplified at the same amplification rate while the tagging sequences are kept tagged through the amplification. At least one of the primers used in PCR amplification is labeled with fluorophore. The fluorophore labeled DNA fragment amplified by PCR are analyzed by gel electrophoresis coupled with fluorescence detection. The DNA fragments originated from different DNA samples appear in the different positions in an electropherogram which is used for the comparative analysis of the gene expression.
ATAC PCR is effective when one target DNA fragment species in different DNA samples is comparatively analyzed. However, when plural of target DNA fragment species in various samples are the targets of comparative analysis, the accurate comparative analysis becomes difficult because unexpected and undesired side reactions frequently occur in a PCR with plural pairs of primers. Various primers in the reaction mixture may interact with DNA fragments other than proper target DNA fragments and may produce unwanted products. This can be overcome by using two types of primers; the first primer is common to all the target DNA fragments and free in a liquid phase, the second primers are specific to the target DNA fragments and is fixed on solid supports. This prevents the interaction between two different specific primers through a PCR reaction. PCR amplification is carried out under the following conditions: the primers specific to the target DNA fragments, respectively, are immobilized on the surfaces of beads or the like so as to be separated on the basis of the kinds of the primers, and the primers having a common base sequence are mobile in a solution. Thus, the production and amplification of undesired DNA fragments other than the target DNA fragments are prevented.
Thus, the target DNA fragment species are mixed and then subjected to PCR simultaneously. The base sequence of the priming site is the same for different DNA fragments, and most of the base sequences subjected to PCR amplification are the same, and the reactions are carried out in one reaction vessel. Therefore, the target DNA fragment species are amplified under the same conditions. Accordingly, the amplification efficiency of the target DNA fragment species is constant so that a quantitative analysis of DNA fragments is possible.
A specific example of analysis requiring quantitative PCR is the above-mentioned cDNA analysis for monitoring gene expression. Sample cDNAs contain various DNA fragments, and information on gene expression as well as gene function is obtained via quantitative analysis of these DNA fragments in various samples. Usually the copy numbers of target DNAs in samples are small, so that measurement is carried out after PCR amplification.
The PCR amplification should be carried out so as to permit quantitative investigation, and the DNA fragments are preferably reacted at the same time in the same reaction vessel. The PCR conditions should not be different for the DNAs. The PCR amplification of a plurality of DNA species at the same time has been attempted. But it is often unsuccessful because of, for example, the production of unexpected PCR products. On the other hand, when the PCR amplification is carried out for each DNA species independently, the analysis is very labor intensive and troublesome. Further, in gene expression profile analysis, when a uniquely expressed DNA fragment is found, it is preferably taken out for precise analysis.
The recovery of such a DNA fragment from the mixed products has not been carried out because of its difficulty.
Such a situation is common to analyses for diagnoses using genes. Quantitative PCR is important in gene diagnosis and gene expression analysis. The quantitative PCR can easily be carried out, for example, when there is only one target DNA species to be processed in order to find out the presence ratio of the target gene in various environments or in various tissues.
As described above, methods such as ATAC PCR invented for solving this problem are disadvantageous in that they do not permit simultaneous analysis for plurality of target DNA fragment species. It has been an important subject to develop a method for quantitative and comparative analysis of a plurality of target DNA fragment species in various DNA samples, or a sample preparation method.
The present invention is intended to provide a sample preparation method and a sample preparation apparatus which solve the above problems. In detail, the present invention is intended to provide a sample preparation method and a sample preparation apparatus, in which mutual interference by primers is avoided, and artificial DNA fragment production by primer extension is reduced, therefore a plurality of target DNA fragments from various DNA samples are amplified by PCR simultaneously in one reaction vessel.
In the sample preparation method of the present invention, although a plurality of target DNA fragment species are amplified in one reaction vessel, mutual interaction of primers is prevented by carrying out the PCR amplification in mutually isolated places for the target DNA fragment species, respectively. Primers (specific primers) hybridizing specifically to the target DNA fragment species, respectively are immobilized on surfaces of fine particles or beads, and target DNA fragment species are amplified by PCR on the surfaces of the corresponding fine particles or beads. Each of the specific primers immobilized on fine particles or beads, and a primer (this primer is referred to as a mobile (or free) primer or a common primer) in the liquid phase are used for complementary strand extension.
In addition, mutual interaction of the primers is prevented by localizing the positions of holding the fine particles or beads in the vessel, depending on the kinds of the specific probes (primers) immobilized on the surfaces of the fine particles or beads. After completion of PCR, the solid supports such as the fine particles or beads, fibers or the like are separated and recovered, and DNA fragment species trapped on the surfaces of the solid supports are also separated and recovered. The specific primers have substantially the same length but have different base sequences according to their target DNA fragment sequences.
In analysis using the sample preparation method of the present invention, the discrimination of DNA target fragments in various DNA samples is made possible by bonding different kinds of oligomers as priming regions to the ends of target DNA fragments, respectively, according to the DNA samples.
As to the recovery of the PCR products separately according to their kinds, fine particles or beads, which can be discriminated from each other by a chemical or physical property, are used. Each distinguishable fine particle or bead immobilizes the specific primers, specific to a target DNA fragment, on the surface to hold the corresponding DNA fragments amplified through PCR. The fine particles or beads having different chemical or physical properties hold the different kinds of DNA fragments (PCR products) on their surface and are separated by the chemical or physical properties. Consequently the different DNA fragment species or DNA fragment groups produced by PCR are recovered separately with the fine particles or beads. The recovered DNA fragments are analyzed by gel electrophoresis or DNA probe array and so on. Of course the DNA fragments recovered from each kind of fine particles or beads contains DNA fragment copies originated from different DNA samples. The presence ratio of the target DNA fragments among the DNA samples is the same as that of the original one as explained above. The DNA fragments originated from different DNA samples can be distinguished by their lengths because the lengths of the oligomers connected to the target DNA fragment termini differ from DNA sample to DNA sample. This permits the quantitative analysis of the target DNA fragment abundance in various DNA samples.
The sample preparation method of the present invention can be utilized also for carrying out simultaneous PCR amplification of various kinds of target DNA fragments in a plurality of DNA samples to be inspected each containing a plurality of target DNA fragments, and for separating the PCR products. That is, specific primers are immobilized on fine particles or beads and the reactions are carried out in one vessel, or the fine particles or beads are located in different compartments on the basis of the kinds of probes and the PCR amplification is carried out for each of target DNA fragments so that mutual interference of primers may be reduced. After the amplification, the PCR products can be separated and recovered on the basis of the kinds of the DNA fragments and can be analyzed. Of course a DNA probe array can be used as the specific primer support instead of beads.
The sample preparation method of the present invention can provide a method which is impossible according to the referenced prior art, i.e., a method for amplifying the number of copies of a plurality of DNA fragment species derived from a plurality of DNAs amplified while keeping the amplified DNA fragment species quantitatively and comparatively analyzable. According to prior arts, the separation and recovery of PCR amplification products of target DNA fragment species require much labor and time and moreover, the separation and recovery are difficult because gel separation cannot be employed when the DNA fragments have the same length. On the other hand, the separation and recovery can easily be carried out in the present invention.
In the sample preparation method of the present invention, when the base sequences of a plurality of amplified DNA fragment species are determined, sample preparation for the plurality of the amplified DNA fragment species is carried out in one lot in one vessel, and the products are separated and collected for each noted DNA fragment species. Then base sequence determination reaction is carried out for each DNA fragment species, and the reaction products are subjected to gel electrophoresis, whereby the base sequences of the plurality of the DNA fragment species can be determined very efficiently.
The characteristics of typical constitutions of the present invention are explained below. The sample preparation method of the present invention comprises a step of amplifying two or more kinds of target DNA fragments by PCR by using each of specific primers which have base sequences complementary to the target DNA fragments to be amplified and are immobilized on the surfaces of one or more mutually separable groups of supports so as to be separated on the basis of the kinds of the complementary base sequences, and a mobile (free) primer presenting in a solution; and a step of separating and recovering the PCR amplification products, as groups each containing one or more kinds of target DNA fragments.
The sample preparation method of the present invention is characterized also by the following. The mobile (free) primer is a common primer that hybridizes with the two or more kinds of the target DNA fragments in common. The common primer hybridizes with the base sequence of an oligonucleotide introduced into the termini of each of the target DNA fragments. The supports immobilizing the specific primers are a plurality of fine particles or beads, which can be distinguished by specific gravity (weight), color, or size. The kinds of the specific primers immobilized on a bead can be known by the specific weights, respectively, or sizes, of the supports, or colors.
Alternatively, the supports are a plurality of fibers, and the specific primers are immobilized near the ends of different fibers so as to be separated on the basis of the kinds of the specific primers. In particular, the supports are a plurality of mutually discriminable fine particles or beads, which are held in a single reaction cell. The supports are separately held in different compartments in a single capillary.
The fine particles immobilizing the primers are separately held in groups through dummy beads or dummy fine particles, which separate a plurality of compartments. The supports are a plurality of fine particles or beads, which can be discriminated as a plurality of groups which can be discriminated on the basis of the difference of any of the sizes of the fine particles or beads, the specific weights of the fine particles or beads, colors given to the fine particles or beads, and the degrees of magnetization of the fine particles or beads.
The sample preparation method of the present invention comprises a step of amplifying a plurality of target DNA fragments by PCR by using each of the specific primers which have, respectively, base sequences complementary to the two or more kinds, respectively, of the DNA fragments to be amplified, are immobilized on the surface of one or more mutually separable groups of supports so as to be separated on the basis of the kinds of the complementary base sequences, and a free primer in a solution; and a step of separating and recovering the PCR amplification products on the basis of the kinds of DNA fragments.
The free primer is a common primer that hybridizes with the two or more kinds of the DNA fragments in common at an oligonucleotide portion introduced into the end of each DNA fragment.
The sample preparation apparatus as another embodiment of the present invention can be made by a holder having a plurality of holes and a vessel having a concavity for accommodating the edge of the holder. Primers specific to the target DNA fragment species (specific primers), respectively, are immobilized on the inner surfaces of the holes, or they are placed in the holes separately on the basis of the kinds of the specific primers after being immobilized on beads. A free primer common to the target DNA fragment species (a common primer) is mobile in the vessel together with a solution and other reagents (reaction substrates and reagents necessary for PCR, such as enzymes).
When the holder having a plurality of holes is immersed in the reaction solution contained in the vessel, the reaction solution enters all the holes uniformly to be subjected to PCR. The use of immobilized primers (specific primers) specific to the DNA fragment species confines the PCR products in the holes. Therefore, by-products caused by the reactions between two or more kinds of the specific primers in the PCR are not produced.
As described above, according to the present invention, a plurality of DNA fragment species contained in each sample to be analyzed can be amplified by PCR under the same conditions at the same time, and the PCR products can be separated and recovered on the basis of the kinds of the target DNA fragment species.
By immobilizing the specific primers on the surfaces of solid supports such as separate fine particles, beads or fibers to separate them spatially from one another, the reaction area in the PCR can be restricted around the surface areas of the solid supports, and it is possible to prevent the production of undesired DNA products by the cross reaction among the specific primers.
Thus, the quantitative and comparative analysis for a plurality of target DNA fragment species in each sample to be analyzed becomes possible. Furthermore, the method of the present invention saves the labor of sample preparation and permits the reduction of reagents for PCR reaction.
The typical constitution of the present invention is outlined below with reference to FIG. 6.
A plurality of DNA fragment species to be amplified are present in a solution as a mixture. Reagents necessary for PCR, such as common primers (free primer), reaction substrates and enzymes are added into the aforesaid solution to obtain a reaction mixture. Primers specific to DNA fragment species (specific primers) to be amplified, respectively, are immobilized on beads, which are placed in the holes 301-1, xcx9c, 301-9 of a holder 302 in distinction from one another on the basis of kinds of the specific primers.
Needless to say, the alternative way of holding specific primers is to immobilize them on the inner surfaces of the holes so as to be separately placed in different holes on the basis of the kinds of the specific primers.
When the holder having a plurality of the holes is immersed in the reaction mixture contained in a vessel, the reaction mixture containing all the target DNA fragment species, the reagent for reaction and the common primer goes into the holes. When PCR is carried out in each hole, the reaction conditions are the same in all the holes and the target DNA fragment species to be amplified are amplified by PCR in compartments, respectively, spatially separated on the basis of the kinds of the target DNA fragments.
The reaction solution can go in and out of the holes freely and the various target DNA fragment species can be amplified under the same conditions without mutual interaction, by the confinement of only the specific primer to the specific places. DNA fragments produced by the amplification in each hole can, of course, be separately collected and can be analyzed.
According to the present invention, mutual interaction of the primers can be avoided, target DNA fragment species in a plurality of samples can be amplified by PCR under the same conditions at the same time, and the PCR products can be separated and recovered on the basis of the kinds of the target DNA fragment species.